Advances in RNA interference (RNAi)-based methodologies and tools in recent years have provided unprecedented opportunities to systematically interrogate the function of all genes relevant to a particular process. In Drosophila, arguably the best-understood multicellular organism and a proven model system for studying human diseases, conditional and tissue-specific expression of RNAi constructs using the Gal4/UAS system has emerged as the method of choice to determine the functions of all genes. To facilitate in vivo RNAi studies, a number of large-scale resources have been developed that rely on long double- stranded RNAs (dsRNAs) as the active RNAi reagents. These long dsRNAs are problematic as they do not work in the female germline and many have poor knockdown efficiencies. This application is to request continued funding for the Transgenic RNAi Project (TRiP; ://www.flyrnai.org) at Harvard Medical School. The goals of the TRiP, funded by NIH/NIGMS R01-GM084947 three years ago, were to improve methods of transgenic RNAi and to generate RNAi lines for the community. During this funding period we have optimized vectors for transgenic RNAi and shown that small hairpin RNAs (shRNAs) are much better reagents than long dsRNAs for in vivo RNAi as they are more effective in somatic tissues and work in both the male and female germlines. Based on the growing need by the community for shRNA fly lines, we have already generated more than 3000 fly lines that are distributed by the Bloomington Drosophila Stock Center. As we move forward, we propose to continue expanding the collection of TRiP shRNA lines (Aim 1), which we will accomplish by generating lines ourselves as well as coordinating the production of lines by a number of outside groups (the Japanese stock center, the Tsinghua University Chinese stock center, and individual laboratories) that are interested in helping build the resource. In addition as information on the efficacy of RNAi lines is currently not being tracked, we will establish the Digital Red Book of RNAi (Aim 2) by collecting the increasing amount of information available on existing lines and performing a number of validation experiments to evaluate the performance of existing and new lines. Well-organized public availability of this information will not only help individual researchers select the best lines for their experiments but also help us identify which additional shRNA lines need to be produced. Together, these Aims will provide the Drosophila community with an eagerly awaited, comprehensive in vivo RNAi resource that will foster studies at the single gene level and facilitate larger-scale screens. This is criticall important as both approaches make it possible for researchers to address fundamental biological questions that will ultimately lead to much-needed understandings of gene function and vital translational projects.